Understanding protein-protein interactions is important for basic research as well as various biomedical and other practical applications. Examples of this kind include binding between peptide fragments or epitopes and antibodies, the interaction between proteins and short fragments of other proteins, for example, MDM2 and p53 transactivation domain, Bcl-xL and Bak peptide, as well as binding between peptides referred to as aptamers to their target proteins. Development of simple and reliable methods of identifying peptide binders for proteins would help to understand the mechanisms of protein-protein interaction and open new opportunities for drug discovery.
State of the art in silico peptide discovery is guided by the X-ray crystal structures and relies on existing structural information. The application of such methods to de novo discovery of peptide binders is limited. To date, experimental methods provided the most effective approaches for peptide discovery. The commonly used methods rely on combinatorial peptide libraries in which peptides are linked to DNA or RNA molecules encoding them. The libraries are panned against immobilized target protein to identify most specific and tight binding peptides. Selection procedure performed in several rounds and after each round selected peptides are identified by PCR amplification of the encoding nucleic acid sequences. Different variations of this approach have been developed and successfully applied to peptide discovery; the most commonly used are phage display, ribosome display, and mRNA-display methods. Despite the unquestionable success of these methods at identifying peptide binders, they are expensive, time consuming and prone to contamination. Furthermore, the existing methods do not ensure that the top selected peptide binders are indeed the best binders and whether they can be improved. Currently, there is no systematic approach to this problem and laborious trial and error optimization techniques are used.
Another powerful experimental method to study peptide-protein interactions are peptide arrays. Peptide arrays could be made off peptides synthesized using solid phase peptide synthesis and then immobilized on solid support or could be directly prepared by in situ synthesis methods. Although peptide arrays are commercially available, their application is limited by a relatively low density and high cost of manufacturing. Both of these issues can be addressed by use of maskless light-directed technology, see (Pellois, Zhou et al. (2002) Individually addressable parallel peptide synthesis on microchips) and U.S. Pat. No. 6,375,903.
Using a MAS instrument, the selection of nucleic acid or peptide sequences to be constructed on the microarray is under software control such that it is now possible to create individually-customized arrays based on the particular needs of an investigator. In general, MAS-based microarray synthesis technology allows for the parallel synthesis of millions of unique oligonucleotide or peptide features in a very small area of a standard microscope slide. The microarrays are generally synthesized by using light to direct which oligonucleotides or peptides are synthesized at specific locations on an array, these locations being called features.
One application of specific peptide binders is medical diagnostics. Prostate cancer is the most commonly diagnosed form of cancer in American men over the age of 50. Currently, the standard for detection of prostate cancer involves screening blood for levels of prostate specific antigen (PSA), digital-rectal examination, and needle biopsy of the prostate. PSA levels, however, may be compromised by variations in the amount of PSA produced by benign prostatic tissue (see, for example, Brawer M K, CA Cancer J Clin 49:264-281 (1999)). Thus, current PSA assays (and PSA alone) are not perfect for identifying prostate cancer (for and distinguishing it from benign hyperplasia. Thus, there is a need to identify means of more specifically targeting PSA and possibly additional biomarkers to improve diagnostic accuracy.
As noted above, the precise detection and identification of biologically relevant molecules within samples of interest is also important in the field of drug discovery. There exists an unmet need for a more efficient and successful method of identifying therapeutic candidates for existing and potential new targets, including targets that are presently considered “undruggable.”